DESCRIPTION: The interaction of viral and host cell factors determines the fate of viral infection. Dr. Azizkhan has been studying the effect of human cytomegalovirus (HCMV) infection and expression of HCMV-immediate early (IE) genes on the cell cycle/growth regulated dihydrofolate reductase (DHFR) gene. The E2F family of transcription factors are involved in basal transcription and cell cycle regulation of the DHFR gene. The E2F binding site is required for trans activation of DHFR by the 72 kDa IE1 gene product (IE72), and E2F-1 and -2 interact with IE72 in cells and in cell-free assay. Further functional and molecular analyses of HCMV IE72 and E2F are proposed to identify HCMV-host cell interactions that regulate viral replication. To this end, they will: (1) characterize the role of IE72 and its E2F-mediated effects in HCMV infection; (2) characterize the effects of IE72 on cell proliferation, cell cycle kinetics and cellular gene expression; and (3) determine the mechanism of activation of E2F-dependent transcription by HCMV IE72. In order to determine the precise role of IE72 in HCMV infection and the associated cellular response, they will determine the effect of viral infection on E2F synthesis and its association with other cellular proteins. To determine whether the interaction of IE72 with E2Fs and the kinase activity of IE72 are essential for viral replication, dominant negative mutants of IE72 that preclude these activities will be used. Experiments to address the second aim utilize a stable cell line with inducible IE72 expression. The effect of IE72 on host cell proliferation and gene expression and the role of host cell growth state in modulating its response to IE72 will be ascertained. Experiments to address the third aim involve establishing an in vitro transcription system to determine whether the effects of IE72 and E2F-dependent transcription. The role of the kinase activity of IE72 and its dissociation of E2F from specific pocket proteins in transcriptional activation will be addressed. In summary, their identification of E2F as a target of HCMV-IE72 directed them to explore the role of E2F as an essential mediator of virus effects. The goal of these experiments is to establish how the effects of HCMV on cellular transcription factors and other factors involved in cell cycle control relate to host cell infectivity and to activation of both cellular and viral DNA synthesis and gene expression.